Three major proteolytic pathways (lysosomal, calcium-dependent, and the ATP-dependent pathways) exist in eukaryotic cells. The ATP-dependent pathway has long been known orchestrate specific degradation of native proteins. Recently it has become clear that the ATP-dependent ubiquitin mediated intracellular pathway is responsible for selective degradation of intact biomolecules as an efficiently evolved mechanism to adapt cellular physiology to the needs of the organism. Proteolysis is a powerful means of regulation due to the speed and irreversibility which enables the cell to rapidly eliminate or reduce the functional level of a particular biological molecule. See, e.g., Jentsch, S., et al., Selective Protein Degradation: A Journey's end Within the Proteasome, Cell, 82:129 (1995). The critical role of ubiquitin-dependent proteolysis has steadily become increasingly clear, for example, in the normal degradation of oncoproteins and tumor suppressers in cell cycle control as well as in stress response and the immune system. Hochstrasser, M., Current Biology, 4:1024 (1992); Deshaies, R. J., Trends Cell Biol., 5:428 (1995); Hilt, W., et al., Trends Biol. Sci., 21:96 (1996).
Ubiquitin is a heat-stable 76-amino acid biomolecule considered to be the most highly conserved protein known. Selective protein degradation via the ubiquitin pathway generally involves tagging of the target protein (substrate) by covalent attachment of multiple molecules of ubiquitin, and degradation of the target by the 26 S proteasome complex. Proteins are marked for direction to the proteasome via the covalent addition of branched polyubiquitin chains to the .alpha.-amino group of one or more surface lysines. The amide linkage of ubiquitin to a substrate protein is generally carried out by three classes of accessory enzymes in a sequential reaction. Ubiquitin activating enzymes (E1) activate ubiquitin by forming a high energy thiol ester intermediate. Activation of the C-terminal Gly of ubiquitin by E1, is followed by the activity of a ubiquitin conjugating enzyme E2 which serves as a carrier of the activated thiol ester form of ubiquitin during the transfer of ubiquitin directly to the third enzyme, E3 ubiquitin protein ligase. E3 ubiquitin protein ligase is responsible for the final step in the conjugation process which results in the formation of an isopeptide bond between the activated Gly residue of ubiquitin, and an (.alpha.-NH group of a Lys residue in the substrate or a previously conjugated ubiquitin moiety. See, e.g., Hochstrasser, M., Ubiquitin-Dependent Protein Degradation, Annu. Rev. Genet., 30:405 (1996).
In a reconstituted system, for example, all three categories of affinity purified enzymes (E1 , E2, and E3) are required for the breakdown of .sup.125 I-albumin to acid-soluble material in the presence of ubiquitin and ATP. Sears, C., et al., NF-kB p105 Processing Via the Ubiquitin-Proteasome Pathway, J Biol Chem., 273:1409 (1998). The high specificity of the ubiquitin selective-destruction pathway is predicted to allow the development of new classes of highly potent and selective low molecular weight enzyme inhibitors targeting particular members of the ubiquitin pathway that control the intracellular levels of a wide range of important regulatory proteins. Rolfe, M., et al., The Ubiquitin-Mediated Proteolytic Pathway as a Therapeutic Area, J. Mol. Med. 75:5-17 (1997).
Compelling evidence has been presented that implicates ubiquitination in the turnover of the tumor supressor protein, p53, cell cycle regulators cyclin A and cyclin B, the kinase c-mos, the cystic fibrosis transmembrane conductance regulator, the DNA repair protein O.sup.6 -methylguanine-DNA methyl transferase, the transcriptional co-activator p300, the transcription factors c-jun, c-fos, I.kappa.B/NF.kappa.B, the transcription factors c-myc, DP1, and E2F, the regulatory subunit of cAMP-dependent protein kinase, receptors for peptide growth factors, estradiol receptor, as well as oncoprotein E1A. Moreover, as a corollary, pharmacological intervention which alters the half-lives of these cellular proteins is expected to have significant value in wide therapeutic potential, particularly in the areas of autoimmune disease, inflammation, cancer, as well as other proliferative disorders. Rolfe, M., et al., The Ubiquitin-Mediated Proteolytic Pathway as a Therapeutic Area, J. Mol. Med., 75:5 (1997).
E3 ubiquitin protein ligase, as the final player in the ubiquitination process, is responsible for target specificity of ubiquitin-dependent proteolysis. A number of E3 ubiquitin-protein ligases have previously been identified. See, e.g., D'Andrea, A. D., et al., Nature Genetics, 18:97 (1998); Gonen, H., et al., Isolation, Characterization, and Purification of a Novel Ubiquitin-Protein Ligase, E3--Targeting of Protein Substrates via Multiple and Distinct Recognition Signals and Conjugating Enzymes, J. Biol. Chem., 271:302 (1996); Scheffner, M., et al., The HPV-16 E6 and E6-AP Complex Functions as a Ubiquitin-Protein Ligase in the Ubiquitination of p53, Cell, 75:495 (1993); Huibregtse, J. M., et al., A Family of Proteins Structurally and Functionally Related to the E6-AP Ubiquitin Protein Ligase, PNAS, 92:2563 (1995); Staub, O., et al., WW Domains of Nedd4 Bind to the Proline-Rich PY Motifs in the Epithelial Na+ Channel Deleted in Liddles Syndrome, EMBO, 15:2371 (1996) [the substrate specificity is determined by the E3 ligase]; Siepmann, T. J., et al., Evidence for Stable, Exchangeable E1/E2/E3 Ubiquitin Conjugation Complexes at Physiological Concentrations, FASEB J., 10:2324 (1996).
Other E3 ligases have been extensively evaluated in S. cerevisiae and in cell-free systems using engineered proteins as test substrates. Weissman, A. M., Regulating Protein Degradation by Ubiquitination, Review Immunology Today, 18(4):189 (1997); Sudakin, V., et al., Mol. Biol. Cell, 6:185 (1995); Stancovski, I., et al., Mol. Cell. Biol., 15:7106 (1995); King, R. W., et al., Cell, 81:279 (1995); Chen, Z. J., et al., Cell, 84:853 (1996); Orian, A., et al., J. Biol. Chem., 170:21707 (1995); Varshavsky, A., et al., Cell, 69:725 (1992); Hershko, A., et al., Annu. Rev. Biochem., 61:761 (1992); Ciechanover, A., Cell, 7:13 (1994).
Perry et al., recently identified a single gene which encodes a murine E3 ubiquitin protein ligase of the Hect family, disruption of which is demonstrated to cause an inflammatory phenotype of the mouse as well as enhanced epithelial and haematopoietic cell growth. Perry, W. L., et al., Nature Genetics, 18:143 (1998). The murine E3 results reported by Perry et al indicate the specific ubiquitin-dependent proteolysis is an important mediator in the immune response as well as haematopoietic cell growth in vivo. Moreover, it is recently set forth that modulators of the E3 ubiquitin protein ligase are likely to have significant therapeutic potential, inter alia, as novel anti-inflammatory agents as well as entities to promote wound-healing. D'Andrea, A. D., et al., Nature Genetics, 18:97 (1998); Perry, W. L., et al., Nature Genetics, 18:143 (1998).
However, the previously reported E3 ubiquitin protein ligase is a murine isolate. The availability of an analogous functional human homolog will be ideal for the identification of compounds which modulate the specific biological activity of the E3 protein ligase and, as a corollary, modulate the physiological conditions associated with aberrant ubiquitin dependent proteolysis in human physiology. The availability of an analogous functional human homolog will also be ideal for the diagnosis, study, prevention, and treatment of pathophysiological disorders related to the biological molecule.